BACKGROUND: Ischemic postconditioning protects the myocardium from ischemia/recursion injury via maintaining 3-minute acidosis initially. But its effect on the skeletal muscle remains unclear. OBJECTIVE: To dynamically measure the pH values in rat skeletal muscle after ischemia, and then to simulate acidic perfusate infusion to investigate the effect of ischemic postconditioning on ischemia/reperfusion injury. METHODS: Based on the ischemia/reperfusion injury model and ischemic postconditioning protocol in previous study, dynamic measurement of pH values in rat skeletal muscle was conducted using pH instrument at the global ischemia, ischemic postconditioning (30/30 seconds) and reperfusion period, and then the acidic perfusate equivalent to pH in ischemic postconditioning period was prepared with lactic acid and normal saline. Twenty-five healthy adult male Sprague-Dawley rats were randomly divided into sham, ischemia/reperfusion, ischemic postconditioning, lactic acid, and normal saline groups (n=5 per group). Blood samples were collected to detect lactate dehydrogenase level. The samples from gastrocnemius were harvested to calculate the wet/dry ratio, level of myeloperoxidase, and infarct size through triphenyltetrazolium chloride staining. The samples from the right tibialis anterior muscle were taken to detect the expression level of Erk1/2 in the MAPK signaling pathway by western blot assay. RESULTS AND CONCLUSION: A prolonged acidic platform was detected in the early reperfusion during ischemic postconditioning, on which the pH value was 6.81±0.133, and the duration was 2 minutes and 40 seconds. The levels of lactate dehydrogenase and myeloperoxidase as well as the wet/dry ratio in the ischemic postconditioning and lactic acid groups were significantly lower than those in the ischemia/reperfusion group (P < 0.05). Western blot assay results showed that the expression level of p-Erk in the ischemic postconditioning, lactic acid and normal saline groups was significantly higher than that in the ischemia/reperfusion group (P < 0.05). Triphenyltetrazolium chloride staining results showed that compared with the ischemia/reperfusion group, the infarct area was significantly reduced in the postconditioning and lactic acid groups (P < 0.05). These findings suggest the existence of a short acidosis during ischemic postconditioning in the early reperfusion, and acidic perfusate can simulate the ischemic postconditioning and effectively attenuate ischemia/reperfusion injury in the rat skeletal muscle via activating Erk1/2 in RISK signaling pathway.

BACKGROUND:Reperfusion injury salvage kinase (RISK) pathway plays an important role in protective mechanism against ischemia reperfusion injury (IRI) induced by both ischemic pre-and post-conditioning. Many researches have been carried out on RISK pathway mechanism underlying ischemic post-conditioning conferring cardioprotection against IRI;however, there is less research about its effect on IRI in the skeletal muscle.
OBJECTIVE:To investigate the protective effect of an optimized protocol of ischemic post-conditioning on IRI in rat skeletal muscle and its underlying mechanism.
METHODS:Eighteen male Sprague-Dawley rats were equivalently randomized into IRI, ischemic post-conditioning and control groups. Rats were given occlusion or disocclusion of the right femoral artery of the right lower limb. Subsequently, the IRI group rats were subjected to 24 hours of reperfusion;the ischemic post-conditioning group immediately given 4 cycles of 30 seconds reperfusion/30 seconds ischemia, followed by 24 hours of reperfusion;the control group given no intervention.
RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that in the ischemic post-conditioning group, the morphology of muscle fibers changed little, with fewer inflammatory lesions and milder edema compared with the IRI group. The infarct size with TTC staining in the ischemic post-conditioning group was smaller than that in the IRI group. Western blot analysis revealed that the expressions of phospho-Akt and phosphorylated endothelial nitric oxide synthase-S1177 were significantly increased, but the expression of phosphorylated type endothelial nitric oxide synthase-Thr495 was much decreased in the ischemic post-conditioning group compared with the IRI group. The measurement of mitochondrial permeability transition pore opening with Ca2+induction showed that the absorbance values in the ischemic post-conditioning group were significantly lower than those in the IRI group (P<0.05). These results indicate that ischemia-reperfusion injury can be improved by applying an optimal protocol of ischemic post-conditioning in rat skeletal muscle. The underlying mechanism may be associated with the activation of RISK signaling pathway to inhibit opening of mitochondrial permeability transition pore, thereby contributing to the enhanced tolerance to IRI in rat skeletal muscle.

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27(Kip1), a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27(Kip1) expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27(Kip1) and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27(Kip1) expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27(Kip1)-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27(Kip1) expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27(Kip1) expression. In contrast, miR-186 expression positively associated with p27(Kip1) expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27(Kip1)-mediated cell cycle alternation.
Animals ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; Genes, Reporter ; Humans ; Luciferases ; Pituitary Neoplasms ; Rats ; Up-Regulation

Postzygotic mutations are acquired in normal tissues throughout an individual's lifetime and hold clues for identifying mutagenic factors.Here,we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals.In blood,sperm,and muscle cells,we resolved three common types of mutational signatures.Signatures A and B represent clock-like mutational processes,and the polymorphisms of epigenetic regulation genes influence the pro-portion of signature B in mutation profiles.Notably,signature C,characterized by C＞T transitions at GpCpN sites,tends to be a feature of diverse normal tissues.Mutations of this type are likely to occur early during embryonic development,supported by their relatively high allelic frequencies,presence in multiple tissues,and decrease in occurrence with age.Almost none of the public datasets for tumors feature this signature,except for 19.6％of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1(HIF-1)signaling pathway.Moreover,the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α.Thus,embryonic hypoxia may explain this novel signature across multiple normal tissues.Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C＞T transitions at GpCpN sites;and indi-viduals'genetic background may also influence their postzygotic mutation profiles.